1. Field of the Invention
The present invention relates to a method of detecting lactic acid bacteria which cause beer turbidity or cloudiness and affect beer quality. The present invention also relates to a protein specific to lactic acid bacteria which cause beer turbidity or cloudiness and a polynucleotide encoding the protein.
2. Description of the Background Art
Beer is a drink that has limited carbon sources, contains alcohol and carbon dioxide gas, presents low pH and anaerobic conditions, and further contains substances having antimicrobial activity derived from hops such as isohumulone, making it difficult for microbial contamination or microbial growth to occur. However, it is known that when, even under these conditions, beer is contaminated with a certain kind of lactic acid bacteria which belong to genus Lactobacillus or genus Pediococcus, the bacteria grow and cause beer turbidity or cloudiness to greatly affect the quality of beer. Typical examples of such lactic acid bacteria include L. brevis, P. damnosus and L. lindneri but some other lactic acid bacteria are also confirmed to have such activity. However, it is known that even among lactic acid bacteria that belong to the same species, some strains grow in beer to cause turbidity and cloudiness (hereinafter referred to as “beer-spoilage lactic acid bacteria”) while others don't grow (hereinafter referred to as “non-beer-spoilage lactic acid bacteria”). This inconsistency often occurs with strains of L. brevis and P. damnosus. Therefore, beer-spoilage lactic acid bacteria cannot be directly detected simply by distinguishing the species.
Methods for the detection and distinction of lactic acid bacteria that affect the quality of beer have been studied to date. In a typical method, DNA is extracted from lactic acid bacteria and the presence of a particular gene of beer-spoilage lactic acid bacteria (hereinafter referred to as a “marker”) is confirmed by Southern hybridization reaction or the like. In particular today, a method of distinguishing lactic acid bacteria by amplifying a DNA sequence by a PCR (polymerase chain reaction) method using an oligonucleotide as a primer (Japanese Patent Laid-Open Publication No. 141899/1994) is used to distinguish lactic acid bacteria. This method has advantages such that only a small amount of bacterial cells is required for distinction, the operation is simple, and a result can be obtained in a short time.
When this method is used for distinguishing beer-spoilage lactic acid bacterial, its success or failure is most influenced by a marker and a primer sequence constructed based on the marker. Namely, beer-spoilage lactic acid bacteria having a marker and a primer sequence constructed from the marker can easily be detected, but beer-spoilage lactic acid bacteria not having such sequences cannot be detected even if they are present. On the other hand, when non-beer-spoilage lactic acid bacteria having such sequences are present, they are mistakenly detected as beer-spoilage lactic acid bacteria.
In the conventional method for distinguishing beer-spoilage lactic acid bacteria by PCR, an attempt has been made to solve the abovementioned problem by using a 16S ribosomal RNA gene as a marker and constructing a primer based on this marker. The 16S ribosomal RNA gene is a gene essential for sustaining bacterial viability and is highly preservable, but it has a region where the DNA sequence can be different in different organic species, which is called a variable region. This variable region is widely used in classification of organic species, genealogical analysis of evolution, and the like, and similarly for lactic acid bacteria, this gene can be used as a marker to detect and distinguish the abovementioned L. brevis, P. damnosus, L. lindneri, and the like.
However, there are two problems with this method. First, it is highly probable that since the DNA sequence of the primer is associated with a gene which is not directly related to the beer-spoilage ability, beer-spoilage lactic acid bacteria having a certain mutation in this site cannot be detected, even if they are present. The variable region of the 16S ribosomal RNA gene is considered to be vulnerable to mutation and thus beer-spoilage lactic acid bacteria having mutation in a small region of the PCR primer may not be detected even if they are present.
The other problem is that this method, which is essentially for the distinction of organic species, cannot be applicable to distinguish beer-spoilage lactic acid bacteria from non-beer-spoilage lactic acid bacteria, particularly for L. brevis and P. damnosus, because among lactic acid bacteria that belong to the same species, some strains could be beer-spoilage lactic acid bacteria while others could be non-beer-spoilage lactic acid bacteria, as mentioned above.
Accordingly, there has been a need for a marker gene which detects beer-spoilage lactic acid bacteria more accurately than the 16S ribosomal RNA gene.
An example of the most desirable marker to detect beer-spoilage lactic acid bacteria more accurately than the 16S ribosomal RNA gene is firstly the very causative gene that renders lactic acid bacteria beer-spoilage ability. Further, the next preferable marker is a base sequence which is known to be located in the proximity to the gene that renders lactic acid bacteria beer-spoilage ability.
There have been several reports on genes which are considered to be important for beer-spoilage lactic acid bacteria to acquire the beer-spoilage ability. For example, there is a report on a method of constructing a probe for the distinction of beer-spoilage lactic acid bacteria from a plasmid which is known to grow in lactic acid bacteria which have become resistant to a high hop concentration by gradual acclimatization to a medium containing a high concentration of hops (Japanese Patent Publication No. 3057552).
However, this method has a problem that it does not necessarily reflect the primary difference between beer-spoilage lactic acid bacteria and non-beer-spoilage lactic acid bacteria since the lactic acid bacteria are treated forcefully to acquire the hop resistance.
There are reports on obtaining genes specific to beer-spoilage lactic acid bacteria. For example, as for genes derived from L. brevis, horA obtained as a hop resistance gene (Journal of the American Society of Brewing Chemistry, 55, 137-140, 1997) and hita, which is considered to be a gene related to manganese intake (Federation of European Microbiological Societies and Netherlands Society for Microbiology, Abstract of the Sixth Lactic Acid Bacteria Symposium, September 1999), have been reported. Though not quite satisfactorily, these gene are considered to be effective as markers for determining beer-spoilage lactic acid bacteria for L. brevis; however, horA erroneously identifies non-beer-spoilage lactic acid bacteria as beer-spoilage lactic acid bacteria at a high frequency for L. brevis and neither of the genes can distinguish beer-spoilage lactic acid bacteria for P. damnosus. Therefore, there has been a need for a marker for the detection of beer-spoilage lactic acid bacteria which is widely applicable and has a high correlation with the spoilage ability as compared to these previously reported markers.